Fluorescence Cross-Correlation Spectroscopy for Real-Time Monitoring of Exogenous DNA Behavior in Living Cells

Abstract

Many kind of DNA transfection techniques have been developed and widely used in the field of biochemical assay (Boussif et al., 1995, Chu et al., 1987, Felgner et al., 1987). Since recent development of fluorescent proteins such as green fluorescent protein (GFP), its variants and bio sensors, DNA transfection techniques play a greater role in the field of cell biology especially in bioimaging studies. DNA transfection is also used as a key technology to gene therapy (Selkirk, 2004, Schaffert et al., 2008). Thus the development of gene delivery vector with high efficiency is expected. Gene delivery systems with artificial nonviral vectors are widely used in the research field of cell biology and also in the clinical field as gene therapy to promote exogenous gene expression or inhibit production of a target protein. Nonviral vectors have advantages such as a low immune response and no risk of a disorder, which might be caused by native viral vector genome integration (Thomas et al., 2003). However, nonviral vectors need to overcome the disadvantage of low gene expression efficiency compared with native viral vectors (Luo et al., 2000). To increase the expression efficiency, it is needed to know gene delivery mechanisms. In general, complexes of exogenous DNAs and a cationic polymer or cationic lipid, commonly used as nonviral vectors, are internalized to target cells by endocytosis. Then a sequential process progresses spontaneously with escape from endosomes, dissociation of complexes, and diffusion of naked DNAs in the cytoplasm to the nucleus (Fig. 1) (Elouahabi et al., 2005). However, such dynamic properties of transfected DNAs are not yet clearly distinguished because of the lack of suitable technique to observe intracellular DNA behavior. Fluorescence correlation spectroscopy (FCS) is a method based on observation of fluorescence intensity fluctuations that is the result of single fluorescent molecules diffuse in and out of small detection area (Fig. 2A, B) (Eigen et al., 1994). FCS technique can be applied for quantification of the absolute number of fluorescently labeled particles and measuring the molecular weight or size with extremely high sensitivity in a small sample volume, and a physical separation procedure is not needed. Therefore, FCS has currently employed to investigate molecular size or interactions in vitro and in vivo (Kinjo et al., 1995, Kitamura et al., 2006, Remaut et al., 2007, Kinjo et al., 2010). Dual color fluorescence cross-correlation spectroscopy (FCCS), an extended technique of FCS, can monitor molecular interactions directly in addition to parameters given from FCS measurements (Saito et al., 2004, Kogure et al., 2006, Bacia et al., 2007, Kim et al., 2007).